What to expect after running installation and do you have any test data to check if it's working?

[MauriAndresMU1313]

Hi team!
I'm back with some questions, I while ago I asked for help to pull the singularity image or solving an issue with SLURM-srun.
HPC policy obligate me to remove any data or libraries every 60 days, so in this case the last time that I installed this tool was more that 60 days, now I'm trying again, however, some changes were made compared with the last version.
So I followed all the steps:
STEP 1:

mamba create -n snake -c conda-forge -c bioconda python=3.12 \
  snakemake snakemake-executor-plugin-slurm snakedeploy pulp click coincbc
mamba activate snake
pip install alphapulldown-input-parser

STEP 2:

snakedeploy deploy-workflow \
  https://github.com/KosinskiLab/AlphaPulldownSnakemake \
  AlphaPulldownSnakemake \
  --tag 2.1.5
cd AlphaPulldownSnakemake

Then, try to execute and see if is working:

snakemake --profile config/profiles/desktop --cores 8

In this case, only getting this output in the terminal:

Using profile config/profiles/desktop for setting default command line arguments.
Building DAG of jobs...
Unlocked working directory.

and in the main directory I only have a new directory: temp_fold (empty). I have the following singularity output in the terminal:

Click to expand

INFO:    Converting OCI blobs to SIF format
INFO:    Starting build...
Copying blob sha256:c52fbc198fabe63023b108cc3b8b961a7ec56b8150ace57d46988719a068743c
Copying blob sha256:372c63e0d269b5a5fa727c7a7ba92d216325488deebd06adc5e5765bc44cc7bb
Copying blob sha256:c4d08fd1e0ab51a248964f41fead88265e6ead77d0224c01b85f8d6670426ec6
Copying blob sha256:8958b478682e9dc71ffd5a908f2a77396e2e81782830635623b0f3818d69d246
Copying blob sha256:42c077c10790d51b6f75c4eb895cbd4da37558f7215b39cbf64c46b288f89bda
Copying blob sha256:f9b6f373cd86acdbb5c441abd8bb605469d53b708293c327a0f5a035b9718f96
Copying blob sha256:c33347f78000f9dd335c9a8a4f4bdd818ac6e4a32484d50064838885ec4aa92f
Copying blob sha256:c488f03e2210fb7be508511f13ab406d8f40f21ed7fe5e2f9a3d3225bc8231f6
Copying blob sha256:a2ce6350cb2358462d6890e73cdb173c5beb86a98459850b5351f95132804623
Copying blob sha256:ee90a670bb374382f65ac9e0ec3fa0f517342e17246de768e91c23a8c976968f
Copying blob sha256:e200dca19a2c8f7bbc9900ead6d5091362aa03911af7e29e9ec1bcb5b9741df8
Copying blob sha256:22ba1df4f27e9985f76ce9c35e9c7b63066743b123c351f7bc1bb02efa05ce26
Copying blob sha256:faa1941d57aab30c5e3b84f3c81eaee18c559586934cc0818848f9715c2f4a2f
Copying blob sha256:82015bf136cb1a8a431c9fc1fb4b22f09ad57a36c5764906b9cfb3d085b2fb3f
Copying blob sha256:4b5c47dc35cb6c5122d00c8b561640d5df677230733a913b42d70f1ace09f6e9
Copying blob sha256:e1da6a9b96819bab395075c9a549bc5562dbb077622b6b2e5cb0e4c5de672fd0
Copying blob sha256:7f0b641f7756531bd4e1b60fa86ca3dadb7cc654196d51fc0a33c80ce7f3c6e8
Copying blob sha256:df45a64779daad23422c5ba219c9ea5d3f0594f6ef74ab803c6ce4808d118e60
Copying blob sha256:6b883a91c22238a5926e09a62127f83a5b87de8f950f0251e9d793dfef804cc6
Copying blob sha256:cd9ec7617d2095c1888907e679ab6d22fa79f18f35396112ff9d8b2b2d0b51c1
Copying blob sha256:ce163394856dfa2b81b7087a98cc3f20129baf08006f67082b70722c059b7f5e
Copying blob sha256:65323128ca62b1a0fadeaa7940448927dc531c889dd8633f698d7d5b444bbcd7
Copying blob sha256:ceb32a9f8c9ef4d91aed88b35a6c589e003a6f893311c707c3c1e7199156feb8
Copying blob sha256:ef828e40ad35213f967b3cefa40294dda7677c8caf522e789d7f49e9f14d396e
Copying blob sha256:087d4bd19c83b5addd04d6cac363e8e4217fa8dc7b85728cd2c4f85382147775
Copying blob sha256:1850ea972b42f77800659caaeb8e630b12921626f515d9c83888d609e5ee2db3
Copying blob sha256:3445bdcd8d404970b8da55805cdbd19d101884f6301999a53049a5a95b513996
Copying blob sha256:9123ff4d5892cd91e34ed50132316330ec9b59209100ae708e7db23620b68b31
Copying blob sha256:ef5e884b2cbf7c61c473627e1ac43b59c9efe16d4ff7d751ab869254b846b0dc
Copying blob sha256:4d43d1e642e2e09fb18d688bda6b3c5d69e5ab9f127f49ded0d5e1673558cfc0
Copying blob sha256:81e090244bf8241b030d89f312e2710c60b39d712af728100e1c42d937ca2730
Copying blob sha256:237d3599996f7776faaf7b65acb09dfb982156ff29e85d61489c5b122123b0b0
Copying blob sha256:c88aea0c1d73ce23f90fdb3f4b81c287ee9ebdf7158540c2056d6e329d3c8c18
Copying blob sha256:f6bf9d03c35dca47ea16640cb848b3c2e3cf22ca7b1f4745fa05cebd075ca968
Copying blob sha256:e38861a625d67c71197d5bec46545ad625ae8ab11aa4ea6fa34d3d2ba14f0daa
Copying config sha256:4427d53b08e50b9ba3a93ea1203a93dd1e2ef74a1998cb0e804736f652bf83e7
Writing manifest to image destination
2025/11/15 18:26:30  info unpack layer: sha256:42c077c10790d51b6f75c4eb895cbd4da37558f7215b39cbf64c46b288f89bda
2025/11/15 18:26:34  warn xattr{etc/gshadow} ignoring ENOTSUP on setxattr "user.rootlesscontainers"
2025/11/15 18:26:34  warn xattr{/group/rmedin6-g00/singularity_cache/build-temp-2263623172/rootfs/etc/gshadow} destination filesystem does not support xattrs, further warnings will be suppressed
2025/11/15 18:28:17  info unpack layer: sha256:f9b6f373cd86acdbb5c441abd8bb605469d53b708293c327a0f5a035b9718f96
2025/11/15 18:28:18  warn xattr{etc/gshadow} ignoring ENOTSUP on setxattr "user.rootlesscontainers"
2025/11/15 18:28:18  warn xattr{/group/rmedin6-g00/singularity_cache/build-temp-2263623172/rootfs/etc/gshadow} destination filesystem does not support xattrs, further warnings will be suppressed
2025/11/15 19:16:18  info unpack layer: sha256:c4d08fd1e0ab51a248964f41fead88265e6ead77d0224c01b85f8d6670426ec6
2025/11/15 19:16:19  info unpack layer: sha256:8958b478682e9dc71ffd5a908f2a77396e2e81782830635623b0f3818d69d246
2025/11/15 19:39:39  info unpack layer: sha256:372c63e0d269b5a5fa727c7a7ba92d216325488deebd06adc5e5765bc44cc7bb
2025/11/15 19:39:39  info unpack layer: sha256:c52fbc198fabe63023b108cc3b8b961a7ec56b8150ace57d46988719a068743c
2025/11/15 19:39:39  info unpack layer: sha256:c33347f78000f9dd335c9a8a4f4bdd818ac6e4a32484d50064838885ec4aa92f
2025/11/15 19:39:39  info unpack layer: sha256:c488f03e2210fb7be508511f13ab406d8f40f21ed7fe5e2f9a3d3225bc8231f6
2025/11/15 19:40:05  info unpack layer: sha256:a2ce6350cb2358462d6890e73cdb173c5beb86a98459850b5351f95132804623
2025/11/15 19:43:54  info unpack layer: sha256:ee90a670bb374382f65ac9e0ec3fa0f517342e17246de768e91c23a8c976968f
2025/11/15 19:57:25  info unpack layer: sha256:e200dca19a2c8f7bbc9900ead6d5091362aa03911af7e29e9ec1bcb5b9741df8
2025/11/15 19:57:26  info unpack layer: sha256:22ba1df4f27e9985f76ce9c35e9c7b63066743b123c351f7bc1bb02efa05ce26
2025/11/15 19:57:26  info unpack layer: sha256:faa1941d57aab30c5e3b84f3c81eaee18c559586934cc0818848f9715c2f4a2f
2025/11/15 19:57:26  info unpack layer: sha256:82015bf136cb1a8a431c9fc1fb4b22f09ad57a36c5764906b9cfb3d085b2fb3f
2025/11/15 19:57:27  info unpack layer: sha256:4b5c47dc35cb6c5122d00c8b561640d5df677230733a913b42d70f1ace09f6e9
2025/11/15 19:57:29  info unpack layer: sha256:e1da6a9b96819bab395075c9a549bc5562dbb077622b6b2e5cb0e4c5de672fd0
2025/11/15 19:57:29  info unpack layer: sha256:7f0b641f7756531bd4e1b60fa86ca3dadb7cc654196d51fc0a33c80ce7f3c6e8
2025/11/15 19:57:29  warn xattr{var/lib/apt/lists/auxfiles} ignoring ENOTSUP on setxattr "user.rootlesscontainers"
2025/11/15 19:57:29  warn xattr{/group/rmedin6-g00/singularity_cache/build-temp-2263623172/rootfs/var/lib/apt/lists/auxfiles} destination filesystem does not support xattrs, further warnings will be suppressed
2025/11/15 19:57:30  info unpack layer: sha256:df45a64779daad23422c5ba219c9ea5d3f0594f6ef74ab803c6ce4808d118e60
2025/11/15 19:58:51  warn xattr{usr/local/share/fonts} ignoring ENOTSUP on setxattr "user.rootlesscontainers"
2025/11/15 19:58:51  warn xattr{/group/rmedin6-g00/singularity_cache/build-temp-2263623172/rootfs/usr/local/share/fonts} destination filesystem does not support xattrs, further warnings will be suppressed
2025/11/15 19:59:08  info unpack layer: sha256:6b883a91c22238a5926e09a62127f83a5b87de8f950f0251e9d793dfef804cc6
2025/11/15 20:06:39  info unpack layer: sha256:cd9ec7617d2095c1888907e679ab6d22fa79f18f35396112ff9d8b2b2d0b51c1
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2025/11/15 20:06:43  info unpack layer: sha256:65323128ca62b1a0fadeaa7940448927dc531c889dd8633f698d7d5b444bbcd7
2025/11/15 20:06:44  info unpack layer: sha256:ceb32a9f8c9ef4d91aed88b35a6c589e003a6f893311c707c3c1e7199156feb8
2025/11/15 20:10:58  info unpack layer: sha256:ef828e40ad35213f967b3cefa40294dda7677c8caf522e789d7f49e9f14d396e
2025/11/15 20:11:26  info unpack layer: sha256:087d4bd19c83b5addd04d6cac363e8e4217fa8dc7b85728cd2c4f85382147775
2025/11/15 20:12:45  info unpack layer: sha256:1850ea972b42f77800659caaeb8e630b12921626f515d9c83888d609e5ee2db3
2025/11/15 20:13:53  info unpack layer: sha256:3445bdcd8d404970b8da55805cdbd19d101884f6301999a53049a5a95b513996
2025/11/15 20:13:57  warn xattr{var/cache/apt/archives/partial} ignoring ENOTSUP on setxattr "user.rootlesscontainers"
2025/11/15 20:13:57  warn xattr{/group/rmedin6-g00/singularity_cache/build-temp-2263623172/rootfs/var/cache/apt/archives/partial} destination filesystem does not support xattrs, further warnings will be suppressed
2025/11/15 20:13:58  info unpack layer: sha256:9123ff4d5892cd91e34ed50132316330ec9b59209100ae708e7db23620b68b31
2025/11/15 20:13:58  info unpack layer: sha256:ef5e884b2cbf7c61c473627e1ac43b59c9efe16d4ff7d751ab869254b846b0dc
2025/11/15 20:13:58  info unpack layer: sha256:4d43d1e642e2e09fb18d688bda6b3c5d69e5ab9f127f49ded0d5e1673558cfc0
2025/11/15 20:13:58  info unpack layer: sha256:81e090244bf8241b030d89f312e2710c60b39d712af728100e1c42d937ca2730
2025/11/15 20:13:58  info unpack layer: sha256:237d3599996f7776faaf7b65acb09dfb982156ff29e85d61489c5b122123b0b0
2025/11/15 20:13:58  info unpack layer: sha256:c88aea0c1d73ce23f90fdb3f4b81c287ee9ebdf7158540c2056d6e329d3c8c18
2025/11/15 20:13:58  info unpack layer: sha256:f6bf9d03c35dca47ea16640cb848b3c2e3cf22ca7b1f4745fa05cebd075ca968
2025/11/15 20:13:58  info unpack layer: sha256:e38861a625d67c71197d5bec46545ad625ae8ab11aa4ea6fa34d3d2ba14f0daa
INFO:    Creating SIF file...

If you are asking yourself, did I try the option (I did too):

screen -S snakemake_session
snakemake \
  --executor slurm \
  --profile config/profiles/slurm \
  --jobs 200 \
  --restart-times 5

After this point, I do not know how to continue, it's like something is missing, maybe some executable or files?

• What am I missing?

• Could you please provide some feedback?

• How can I check if it's working?

• Is it possible to provide an example of what to expect after the installation (showing how must look like the directory or the new files pulled)?

Looking forward to your feedback!

Thank you!

[DimaMolod]

Hi @MauriAndresMU1313!
after snakedeploy, you will get structure like this:

|-- config
|   |-- config.yaml
|   |-- profiles
|   |   |-- desktop
|   |       `-- config.yaml
|   |   |-- slurm
|   |       `-- config.yaml
|   |-- sample_sheet.csv
|   `-- split_fasta.py
`-- workflow
    `-- Snakefile

including the configurations file AlphaPulldownSnakemake/config/config.yaml where you need to specify whether you want to use alphafold2 or alphafold3 (for features and predictions), add paths to your AF databases on HPC, alphafold weights, output directory, csv file with complexes to be modeled, etc.
The version of config.yaml you get is configured for EMBL cluster, which will not work out-of-the-box for other clusters.
To check that you configured your run correctly, you can run dry run with
snakemake -n
after running AlphaFold2 (for AF3 layout is slightly different), you'll see output dir like that:

|-- output_directory
|   |-- data
|   |   |-- P01258.fasta
|   |   `-- P01579.fasta
|   |-- features
|   |   |-- P01258_feature_metadata_2025-11-17.json.xz
|   |   |-- P01258.pkl.xz
|   |   |-- P01579_feature_metadata_2025-11-17.json.xz
|   |   `-- P01579.pkl.xz
|   `-- predictions
|       |-- P01258+P01579
|       |-- ...
|-- slurm-logs
|   |-- ...
|   |-- reports
|       `-- all_interfaces.csv

Please refer to https://github.com/KosinskiLab/AlphaPulldownSnakemake/tree/main and snakemake documentation for more details.

[MauriAndresMU1313]

Thank you for provide that feedback! My bad for the basic questions...

Now looks like is finally working, at least in the dry-run option. I'm using the test data specified on the sample sheet file. In this case, I'm having the following error:

/bin/bash: line 3: create_individual_features.py: command not found

Click to expand

/bin/bash: line 3: create_individual_features.py: command not found
RuleException:
CalledProcessError in file "https://raw.githubusercontent.com/KosinskiLab/AlphaPulldownSnakemake/2.1.5/workflow/Snakefile", line 205:
Command ' singularity  exec --home '/scratch/mmora30/workspace/AlphaPulldownSnakemake' --nv --bind '/users/mmora30/.cache/snakemake/snakemake/source-cache' /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/fold_analysis_latest.sif bash -c 'set -euo pipefail;
        create_individual_features.py \
            --fasta_paths=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/data/P01258.fasta \
            --data_dir=/users/mmora30/rmedin6-g00/full_db \
            --output_dir=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/features \
            --save_msa_files=False --use_precomputed_msas=True --max_template_date=2050-01-01 --compress_features=True --data_pipeline=alphafold2'' returned non-zero exit status 127.
  File "https://raw.githubusercontent.com/KosinskiLab/AlphaPulldownSnakemake/2.1.5/workflow/Snakefile", line 205, in __rule_create_features
[Fri Nov 21 16:36:57 2025]
Error in rule create_features:
    message: None
    jobid: 4
    input: /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/data/P01258.fasta
    output: /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/features/P01258.pkl.xz
    shell:

        create_individual_features.py \
            --fasta_paths=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/data/P01258.fasta \
            --data_dir=/users/mmora30/rmedin6-g00/full_db \
            --output_dir=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/features \
            --save_msa_files=False --use_precomputed_msas=True --max_template_date=2050-01-01 --compress_features=True --data_pipeline=alphafold2

        (command exited with non-zero exit code)
Exiting because a job execution failed. Look below for error messages
[Fri Nov 21 16:36:57 2025]
Error in rule create_features:
    message: None
    jobid: 4
    input: /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/data/P01258.fasta
    output: /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/features/P01258.pkl.xz
    shell:

        create_individual_features.py \
            --fasta_paths=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/data/P01258.fasta \
            --data_dir=/users/mmora30/rmedin6-g00/full_db \
            --output_dir=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/features \
            --save_msa_files=False --use_precomputed_msas=True --max_template_date=2050-01-01 --compress_features=True --data_pipeline=alphafold2

        (command exited with non-zero exit code)
[Fri Nov 21 16:36:57 2025]
Error in rule create_features:
    message: None
    jobid: 6
    input: /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/data/P01579.fasta
    output: /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/features/P01579.pkl.xz
    shell:

        create_individual_features.py \
            --fasta_paths=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/data/P01579.fasta \
            --data_dir=/users/mmora30/rmedin6-g00/full_db \
            --output_dir=/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs/features \
            --save_msa_files=False --use_precomputed_msas=True --max_template_date=2050-01-01 --compress_features=True --data_pipeline=alphafold2

        (command exited with non-zero exit code)
Complete log(s): /scratch/mmora30/workspace/AlphaPulldownSnakemake/.snakemake/log/2025-11-21T163656.396070.snakemake.log
WorkflowError:
At least one job did not complete successfully.

I did not find similar error from another user, apparently this line is critical:

--save_msa_files=False --use_precomputed_msas=True --max_template_date=2050-01-01 --compress_features=True --data_pipeline=alphafold2'' returned non-zero exit status 127.

Could you provide some feedback? Looks like is something related to MSA process, however, I checked the create_individual_features.py script, but in the line 3 I did not find relevant information.

Do you need that I provide more information?

Thank you again for your feedback and help!!

[MauriAndresMU1313]

Looking forward to your feedback @DimaMolod
Thank you again!

[DimaMolod]

Hi @MauriAndresMU1313
the problem that you mixed up containers in you config.yaml file and try to use container for analysis pipeline for predictions.
Please check your config.yaml carefully and use the latest version.
BR
Dima

[MauriAndresMU1313]

Wow! Thank you for the quick response.
Let me share with you the config.yaml file:

# List of input sample sheets
input_files:
  - config/sample_sheet.csv
#  - config/sample_sheet.csv

# Delimiter used in protein names
protein_delimiter: "+"

# Whether to drop equivalent permutations (AB and BA are the same) when generating fold specs
exclude_permutations: true

# Directory where all output files will be stored
output_directory: /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/outputs

# Path to AlphaFold databases and to backend weights
# Note prior to version 2.0.4 these were called alphafold_data_directory
databases_directory: /users/mmora30/rmedin6-g00/full_db
backend_weights_directory : /users/mmora30/rmedin6-g00/full_db/params

# Directories containing precomputed features
feature_directory:
  - "/path/to/directory/with/features1"
  - "/path/to/directory/with/features2"

# If True, only generate features without running structure prediction
only_generate_features: false

# Toggle structure analysis after prediction
enable_structure_analysis: true

# Generate an aggregated CSV (reports/all_interfaces.csv) across all folds
generate_recursive_report: true

# Extra CLI flags to pass to alphajudge during recursive report generation
recursive_report_arguments: {}

# Arguments for feature generation
create_feature_arguments:
  --save_msa_files: false  # Do not save multiple sequence alignment (MSA) files
  --use_precomputed_msas: true  # Use precomputed MSA files if available
  --max_template_date: 2050-01-01  # Set maximum template date to include all templates
  --compress_features: true  # Compress generated features
  --data_pipeline: alphafold2 # Use alphafold2 or alphafold3 data pipeline for generating features

# Arguments for structure inference
structure_inference_arguments:
  --fold_backend: alphafold3 # Options: alphafold2, alphafold3, alphalink

# Arguments for structure analysis
analyze_structure_arguments:
  --contact_thresh: 8.0 # Contact threshold for structure analysis
  --pae_filter: 100.0 # PAE filter for structure analysis
  --models_to_analyse: "all"  # Models to analyze

# Memory allocation settings for feature creation and structure inference
feature_create_ram_bytes: 64000
feature_create_ram_scaling: 1.1
structure_inference_ram_bytes: 64000

# Number of threads for AlphaFold inference
alphafold_inference_threads: 8

# SLURM resources
slurm_partition: "gpu"
slurm_gres: "gpu:1"
slurm_qos: "normal"

# Specify the backend by changing the prediction container
# (you can also use local singularity .sif files)
# - "docker://kosinskilab/fold:2.1.2" for AlphaFold2
# - "docker://kosinskilab/alphafold3:2.1.2" for AlphaFold3
# - "docker://kosinskilab/alphalink:2.1.2" for AlphaLink2
# - "/path/to/my/container.sif"
prediction_container: "/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/fold_analysis_latest.sif"

#"docker://kosinskilab/alphafold3:latest"

# Container for structure analysis
analysis_container: "/users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/alphajudge.sif"

So far I downloaded the latest containers, here you can see:

fold_analysis_latest.sif
-rwxr-xr-x.  1 mmora30 HyPER-LG-PROD-Sec 7667847168 Nov 21 00:57 fold_analysis_with_ccp4.sif
drwxr-sr-x.  4 mmora30 HyPER-LG-PROD-Sec      25600 Nov 21 15:27 features_input
-rwxr-xr-x.  1 mmora30 HyPER-LG-PROD-Sec  101879808 Nov 21 15:50 alphajudge.sif

All the process to obtain the container:

# Pull the Singularity image
singularity pull docker://kosinskilab/fold_analysis:latest

singularity build --sandbox ap_sandbox fold_analysis_latest.sif

tar xvzf ccp4-9.0.011-linux64.tar.gz

cp ccp4-9/bin/pisa ccp4-9/bin/sc /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/ap_sandbox/software

cp -rn lib/* /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/ap_sandbox/software/lib64/

singularity build fold_analysis_with_ccp4.sif /users/mmora30/mmora30/workspace/AlphaPulldownSnakemake/ap_sandbox

singularity pull alphajudge.sif docker://kosinskilab/alphajudge:latest

Could you provide some feedback to know which container could be the source of the issue?

Do you see something unusual?

Do you need that I provide more details to make the process smoothly?

Thank you again!